The present invention relates to a novel method for measuring the concentration of glycated hemoglobin (gHb) and the percent hemoglobin A1c (HbA1c) in blood without using antibodies, and more particularly, to a one-read method which does not require an additional measurement of total hemoglobin for assessing percentage hemoglobin A1c 
The importance of diagnosis and monitoring of diabetes is emphasized by the American Diabetes Association reporting that 15.7 million Americans have diabetes, or 5.9% of the population. However, routinely used direct measurement of blood glucose level in patients has a limited value since it gives information only about the glucose concentration at the time of sampling and is influenced dramatically by diet. Nevertheless, an accurate index of a person""s mean blood glucose level over 2 to 3 months can be provided by measurement of a specific type of glycated hemoglobin called the hemoglobin A1c concentration in blood [The Diabetes Control and Complications Trial Research Group, N. Engl. J. Med., 329, 977-986 (1993)]. Glycated hemoglobin, of which about 60% is represented by HbA1c, is formed via nonenzymatic attachment of glucose to the hemoglobin molecule at a rate that is directly proportional to the ambient glucose concentration [Bunn H. F., Haney D. N., Gabbay K. H., Gallop P. N., Biochem. Biophys. Res. Commun., 67, 103-9 (1975) ].In the uncontrolled diabetic, the proportion of HbA,, may be increased three to four fold. For example, a healthy person may have an HbA1c concentration of 4.1-6.5% of the total hemoglobin, whereas in the diabetic the concentration may be up to 20%. Therefore, HbA,, measurement can provide diabetic patients an overview of their success in meeting long-term goals for controlling their blood glucose levels.
A variety of methods have been proposed for measuring HbA,, concentration in a sample of a patient""s blood. They can be broadly divided into two categories on the basis of the principle used to separate glycated from unglycated hemoglobin components:
a) charge differences as in ion-exchange chromatography [U.S. Pat. No. 4,407,961, U.S. Pat. No. 4,649,122, U.S. Pat. No. 4,270,921, U.S. Pat. No. 4,389,491, U.S. Pat. No. 4,436,820] and in electrophoresis [U.S. Pat. No. 4,351,711]. Ion-exchange chromatography involves separation of hemoglobin fractions in microcolumns of ion exchange resin. Glycated hemoglobin elutes first while the non-glycated hemoglobin remains attached to the resin and can be removed by changing the eluting buffer. For proper separation, the composition, pH and ionic strength of the eluting buffer must be maintained within narrow limits. Additionally, the temperature control is critical. Subsequent spectrophotometrical measurement provides the amount of the fraction constituting the glycated hemoglobin. Electrophoresis exploits relative mobility of the hemoglobin fractions in a specially prepared agar medium in an electric field. Common drawbacks for these methods are poor reproducibility, sensitivity to variations in temperature, pH, ionic strength, and sample storage conditions. Also they require expensive equipment and usually prove too slow and cumbersome for practical use.
b) structural characteristics of the carbohydrate groups on the hemoglobin, as in immunoassay [U.S. Pat. No. 4,247,533, U.S. Pat. No. 4,970,171, U.S. Pat. No. 5,206,144] and boronate affinity chromatography [U.S. Pat. No. 4,269,605]. The major limitation of the immunoassay approach is the requirement of an additional independent measurement of the total hemoglobin in order to express the glycated hemoglobin level as a percentage number. Additionally, both immunoassay and high performance chromatography require very expensive reagents and equipment.
The approach based on the use of boronic acid derivatives as affinity matrices for HbA1c isolation is free from limitations that are characteristic for ion-exchange chromatography and electrophoresis and is the most widely accepted method. The representative example of the approach has been disclosed in International Publication No. WO 98/40750 and U.S. Pat. No. 6,162,645. Lee et al. proposed to measure percent of glycated hemoglobin using a single determination that includes incubation of a lysed whole blood sample (i.e. all the red blood cells in the sample has been ruptured) with magnetic microparticles that is coupled with boronic acid. The particles were then attracted to a magnet, washed, and a labeled antibody to human hemoglobin was added and the resulting signal was directly proportional to the %HbA1c in the sample. As it was mentioned above, the need of using antibody makes the method rather expensive.
Sundrehagen, in U.S. Pat. No. 5,919,708, proposed a four step assay for glycated hemoglobin which includes mixing a whole blood sample with a reagent containing agents that lyse erythrocytes, precipitate hemoglobin specifically and bind gHb by means of boronic acid conjugate with a blue dye. Then precipitated hemoglobin, conjugate-bound and unbound, is separated by filtration and washed to remove an excess of colored conjugate. The precipitate is evaluated by measuring the blue (gHb) and the red (total Hb) color intensity and the percentage of A1c is calculated. However this technique suffers from rather complex procedure which requires several manual operations.
Accordingly there is need to develop a simple, inexpensive non-antibody based method of detecting the amount of glycated hemoglobin in a blood sample using a single determination.
An object of the invention is to provide a method and a system for separating glycated hemoglobin from non-glycated hemoglobin, and a method for one-read quantitative determination of the glycated hemoglobin and %HbA,, that does not require an additional measurement of total hemoglobin.
The object of the invention can be achieved by exploiting of two characteristic features of glycated hemoglobin (gHb):
a) Structural Characteristic of the Carbohydrate Group.
The separation of glycated hemoglobin from a blood sample is based on the ability of boronic acids to form cyclic esters with 1,2-cis-diols presented in the glucose moiety of HbA1c molecule (FIG. 1). The isolation is carried out by diluting and lysing a blood sample with a buffer (pH 8-9) containing lysing agent and incubating the solution with boronic acid derivative (e.g., phenylboronic acid) immobilized onto a solid support. After incubation, the unbound hemoglobin is washed away with appropriate buffer.
b) Pseudo-peroxidative Properties of Hemoglobin.
Hemoglobin is known to exhibit pseudo-peroxidase activity (i.e. has catalytic ability like an enzyme such as peroxidase). Such activity can be measured by means of a material which undergoes a detectable change, generally a color change, in the presence of inorganic (e.g., hydrogen peroxide) or organic peroxides. There are many compositions which can be used for this determination, including mono- and diamines, phenols, polyphenols, leucodyes, and other compounds or two component systems which produce colors under the conditions of the assay. of the assay. (FIG. 2).